Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1984 Dec 20;3(13):3323–3328. doi: 10.1002/j.1460-2075.1984.tb02298.x

Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

MJ Daniels 1, CE Barber 1, PC Turner 1, MK Sawczyc 1, RJW Byrde 1, AH Fielding 1
PMCID: PMC557857  PMID: 16453595

Abstract

A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.

Keywords: cloning, cosmid, pathogenicity, Xanthomonas

Full text

PDF
3326

Images in this article

3325Fig. 2.
on p.3325

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. Burkardt H. J., Riess G., Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. doi: 10.1099/00221287-114-2-341. [DOI] [PubMed] [Google Scholar]
  4. Clarke L., Carbon J. Selection of specific clones from colony banks by suppression or complementation tests. Methods Enzymol. 1979;68:396–408. doi: 10.1016/0076-6879(79)68029-1. [DOI] [PubMed] [Google Scholar]
  5. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Daniels M. J. Molecular biology of bacterial plant pathogens. Microbiol Sci. 1984 May;1(2):33–36. [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  10. Grosveld F. G., Dahl H. H., de Boer E., Flavell R. A. Isolation of beta-globin-related genes from a human cosmid library. Gene. 1981 Apr;13(3):227–237. doi: 10.1016/0378-1119(81)90028-7. [DOI] [PubMed] [Google Scholar]
  11. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  12. Kieser T. DNAGEL: a computer program for determining DNA fragment sizes using a small computer equipped with a graphics tablet. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):679–688. doi: 10.1093/nar/12.1part2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Merrick M., Filser M., Kennedy C., Dixon R. Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae. Mol Gen Genet. 1978 Sep 20;165(1):103–111. doi: 10.1007/BF00270382. [DOI] [PubMed] [Google Scholar]
  14. Moores J. C., Magazin M., Ditta G. S., Leong J. Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain. J Bacteriol. 1984 Jan;157(1):53–58. doi: 10.1128/jb.157.1.53-58.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Murray N. E., Brammar W. J., Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. doi: 10.1007/BF02425325. [DOI] [PubMed] [Google Scholar]
  16. Nasuno S., Starr M. P. Polygalacturonic acid trans-eliminase of Xanthomonas campestris. Biochem J. 1967 Jul;104(1):178–185. doi: 10.1042/bj1040178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shaw K. J., Berg C. M. Escherichia coli K-12 auxotrophs induced by insertion of the transposable element Tn5. Genetics. 1979 Jul;92(3):741–747. doi: 10.1093/genetics/92.3.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Starr M. P., Jenkins C. L., Bussey L. B., Andrewes A. G. Chemotaxonomic significance of the xanthomonadins, novel brominated aryl-polyene pigments produced by bacteria of the genus Xanthomonas. Arch Microbiol. 1977 May 13;113(1-2):1–9. doi: 10.1007/BF00428572. [DOI] [PubMed] [Google Scholar]
  19. Story D. L., Shrader R. E., Theriault L. L., Lumijarvi D. L., Shenoy T. S., Savaiano D. A., Shaffer R. H., Ho C. Y., Clifford A. J. Effects of dietary protein, adenine, and allopurinol on growth and metabolism of rats. J Nutr. 1977 Jun;107(6):1044–1052. doi: 10.1093/jn/107.6.1044. [DOI] [PubMed] [Google Scholar]
  20. Wretlind B., Pavlovskis O. R. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol. 1984 Jun;158(3):801–808. doi: 10.1128/jb.158.3.801-808.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES